CN114854674A

CN114854674A - Application of dbcAMP in preparation of primordial follicle in-vitro activator

Info

Publication number: CN114854674A
Application number: CN202210697751.8A
Authority: CN
Inventors: 夏国良; 王超; 郑文影; 张拓
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2022-08-05

Abstract

The invention relates to the technical field of biology, in particular to application of dbcAMP in preparation of an primordial follicle in-vitro activator. The invention provides application of a small molecule drug dbcAMP in preparation of a primordial follicle activation preparation. Experiments show that dbcAMP can increase the phosphorylation level of key protein on the PI3K/mTOR signaling pathway when a mouse ovary is cultured in vitro, thereby amplifying the action of the signaling cascade pathway and further promoting the activation of primordial follicles. Meanwhile, in vivo in situ ovarian injection of dbcAMP can also stimulate primordial follicle activation.

Description

Application of dbcAMP in preparation of primordial follicle in-vitro activator

Technical Field

The invention relates to the technical field of biology, in particular to application of dbcAMP in preparation of an primordial follicle in-vitro activator.

Background

The assisted reproduction technology is the most effective pregnancy-assisting means at the present stage. A prerequisite for traditional assisted reproduction techniques is a mature, healthy oocyte, which requires the presence of a growing follicle in the ovary of a female patient that is capable of responding to gonadotropins. However, premature ovarian failure, polycystic ovarian syndrome, and the development of cancer followed by radiotherapy/chemotherapy do not have enough follicles growing in ovaries to respond to gonadotropins, and it is difficult to obtain mature oocytes.

To solve this problem, conventional assisted reproductive techniques must be improved. In fact, the ovary of the part of patients still has a certain number of primordial follicle resources, so that whether the part of primordial follicles can be utilized to help the part of primordial follicles to grow offspring is a concern.

In recent years, with the help of models such as transgenic mice, a plurality of studies prove that two cell pathways play a key role in primordial follicle activation, namely a PTEN-PI3K/AKT signal pathway in an oocyte and a mTORC1 signal pathway in a propranular cell play a key role in the activation process. Based on the existing mechanism related to primordial follicle activation, through clinical transformation application research, a new in vitro assisted reproduction technology is created, namely: primordial follicle in vitro activation technology (abbreviated in vitro activation, IVA).

The technique that the IVA activates the primordial follicles in vitro by an effective means by using a medicament and carries out the traditional assisted reproduction after the primordial follicles grow to a stage capable of responding to hormones successfully helps part of patients with infertility to successfully breed the next generation. However, in the existing IVA clinical strategy, the safety and success rate of related medicines are still a great hidden trouble, and meanwhile, patients need to undergo multiple laparoscopic surgeries, so that the surgery time is long, and the pain of the patients is increased. Therefore, it is very important to develop a more efficient primordial follicle in vitro activation system.

Disclosure of Invention

In view of this, the invention provides an application of dbcAMP in preparing an in vitro primordial follicle activator, wherein the dbcAMP can increase the phosphorylation level of key proteins on the PI3K/mTOR signaling pathway, and further promote the activation of primordial follicles.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of dbcAMP in preparation of mTOR agonists and/or PI3K/AKT signal pathway key protein agonists.

The invention also provides application of dbcAMP in preparation of a preparation for improving phosphorylation level of mTOR and/or PI3K/AKT signal pathway key proteins.

In some embodiments of the invention, the mTOR and/or PI3K/AKT signaling pathway key protein in the above-described use is derived from ovary.

The invention also provides the application of dbcAMP in preparing the medicine for in vitro primordial follicle activation.

The invention also provides application of dbcAMP in preparation of a medicine for promoting conversion of primordial follicles to primary follicles.

The invention also provides the use of dbcAMP in the manufacture of a medicament for assisted reproduction, including assisted pregnancy.

The invention also provides a medicament for in vitro primordial follicle activation, which comprises dbcAMP and acceptable auxiliary materials and/or auxiliary agents.

In some embodiments of the present invention, the adjuvant and/or auxiliary in the above-mentioned medicament comprises Matrigel.

In some embodiments of the present invention, the content of dbcAMP in the above drug comprises 1. mu. mol/L to 10. mu. mol/L.

In some embodiments of the present invention, the dbcAMP content in the above drug is 10. mu. mol/L.

In some embodiments of the present invention, the adjuvant and/or auxiliary agent in the above-mentioned medicament comprises a binder, a filler, a disintegrant, a lubricant, an isotonicity adjusting agent, a solubilizer, a cosolvent, a preservative, a colorant, a suspending agent, a wetting agent, an emulsifier and/or a surfactant.

The invention also provides a method for activating primordial follicles, and the medicine is injected into ovaries.

In some embodiments of the invention, the method for primordial follicle activation further comprises injecting the drug into the ovarian follicle.

The invention also provides an in-vitro primordial follicle activation method, ovarian tissues are taken to be cultured in an in-vitro activation reagent;

the in vitro activating agent comprises dbcAMP.

The invention has the following effects:

1. the addition of dbcAMP to the ovaries of mice cultured in vitro promotes the activation of primordial follicles present therein. The research shows that the phosphorylation forms of the key protein levels of mTOR, PI3K/AKT signal pathway in the ovary tissues of mice are improved, but the total protein of the mouse is not changed; in the process of adding dbcAMP to culture the ovary of a newborn mouse, the total number of follicles is unchanged, but the number of activated follicles is increased;

2.dbcAMP promotes primordial follicle activation in vivo by in situ ovarian injection without affecting the total number of follicles.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows that the ovaries of newborn 2-day-old mice in the present invention are supplemented with 10. mu. mol/L dbcAMP, and samples are collected one day later to detect the expression of the key proteins in PI3K/AKT and mTOR signaling pathways; wherein the total protein of the key protein is to the left; the key protein is phosphorylated on the right; "Control" is a Control group without added dbcAMP; the results show that: after the in vitro culture of dbcAMP, the result of the ovary of a newborn mouse finds that dbcAMP does not affect the expression amount of total protein of molecules, but leads to the significant up-regulation of the active form of dbcAMP, and the result indicates that the dbcAMP promotes the activation of primordial follicles to be caused by the activation of PI3K/AKT and mTOR pathways;

FIG. 2 shows the effect of the addition of dbcAMP to culture follicles in ovaries of neonatal mice in accordance with the present invention; wherein A is ovarian tissue section; b is the influence of dbcAMP culture for 5 days on the total number of follicles and the number of activated follicles; c is the effect of dbcAMP on total number of follicles at

days

3, 5, and 7; d is the effect of dbcAMP on the follicular activation rate at

days

3, 5, and 7; research shows that the total number of follicles of a new mouse cultured by adding dbcAMP has no obvious difference but the number of growing follicles is increased, which indicates that the ovary of the mouse cultured by adding dbcAMP can promote primordial follicle activation; counting results of 3 days, 5 days and 7 days of in vitro culture of the ovary of the newborn mouse respectively, wherein the results are found as follows: dbcAMP significantly promotes primordial follicle activation, but does not affect the total number of follicles in the ovary; a scale: 50 μm;

FIG. 3 shows that two weeks after 23-day-old mice in the present example were injected with dbcAMP in situ, ovarian tissues were collected for tissue section staining and the number of follicles at each stage was counted; wherein A is ovarian tissue section; b is the effect of dbcAMP on total number of follicles in vivo; c is the influence of dbcAMP on the number of follicles growing in vivo; the results show that: dbcAMP also significantly promotes primordial follicle activation in vivo without affecting the total number of follicles; a scale: 100 μm.

Detailed Description

The invention discloses application of dbcAMP in preparation of an in vitro primordial follicle activator, and a person skilled in the art can realize the application by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Much attention is now paid to the role of cAMP in the female reproductive system in relation to the meiotic progression of oocytes. High levels of cAMP in the cytoplasm of oocytes in growing follicles maintain the first meiosis of oocytes in a blocked state for a long period of time, and oocytes can resume meiosis and maturation only when cAMP synthesis is down-regulated or it is degraded. Studies have shown that the cAMP analog dbcAMP inhibits meiotic recovery from oocytes in lumenal follicles, i.e. inhibits oocyte maturation. Furthermore, elevated cAMP levels are required at early oocyte entry into the meiotic first division, and altering cAMP levels in vitro with drugs can affect primordial follicle bank formation. Since the physiological development process of the follicle has clear temporal and spatial specificity, the primordial follicle activation process may have a distinct physiological regulatory mechanism from the maturation of oocytes and the formation of primordial follicles. Before the present invention has been carried out in connection with studies, it was not clear whether cAMP had an effect on primordial follicle activation in mice.

The invention aims to solve the technical problem of providing the application of dbcAMP in preparing the primordial follicle in-vitro activator. The research shows that when dbcAMP is added in the process of culturing the ovary of a newborn mouse, the total number of follicles is unchanged, and the number of primary follicles is increased, which indicates that part of primordial follicles in the ovary are activated and converted into the primary follicles.

The invention provides application of dbcAMP in preparation of a preparation for activating mTOR.

mtor (mammalian target of rapamycin) is a conserved tyrosine kinase that plays a key role in regulating cell proliferation, growth, survival, division, motility, and angiogenesis. In the invention, the mTOR is mTOR in ovary. The present study shows that dbcAMP treatment can increase the level of phosphorylated mTOR in mouse ovaries. Namely, the invention provides the application of dbcAMP in the preparation of a preparation for increasing the level of phosphorylated mTOR.

The invention also provides application of dbcAMP in preparation of a preparation for activating key proteins on a PI3K/AKT signal pathway.

PI3K is a serine/threonine protein kinase, and regulates various biological processes such as cell metabolism, cell proliferation, apoptosis, cell cycle and the like mainly by activating the activity of AKT. In the invention, the PI3K/AKT signal pathway is a PI3K/AKT signal pathway in ovary. The present study shows that dbcAMP treatment can increase the levels of phosphorylated forms of PI3K/AKT signaling pathway key proteins (AKT, FOXO3a, rpS6) in the mouse ovary. Namely, the invention provides the application of dbcAMP in preparing a preparation for increasing the level of key proteins of a phosphorylated PI3K/AKT signal pathway.

The invention also provides the application of dbcAMP in the preparation of in vitro activators of primordial follicles.

The primordial follicle of the invention is a mouse primordial follicle. The research shows that dbcAMP has an agonistic effect on mTOR and AKT in ovary and has a promoting effect on the transformation of primordial follicle to primary follicle in mouse ovary under the in vitro culture condition. In other words, dbcAMP can act as an in vitro activator of primordial follicles.

The present invention provides an in vitro activating preparation of primordial follicles comprising dbcAMP.

In the preparation provided by the invention, the concentration of dbcAMP is 1 to 10 mu mol/L.

In some embodiments, the concentration of dbcAMP is 10 μmol/L.

In the present invention, the formulation further comprises an ovarian basal medium and a phase transition biogel (Matrigel). Every 100mL of the ovary basal medium consists of water and 0.5g of DMEM, F120.53g, 0.357g of HEPES and 0.357g of NaHCO ₃ 0.2438g, penicillin 0.006g and streptomycin 0.005 g.

The method for in vitro activation of primordial follicles provided by the invention comprises culturing ovarian tissues in the in vitro activating reagent. Specifically, the method comprises the following steps: preheating the in-vitro activating reagent at 37 ℃, separating the ovary of the newborn mouse cleanly in a sterile environment, transferring the separated ovary of the newborn mouse into a preheated activating culture solution, culturing for 1-5 days, and changing the solution every other day.

The in vivo activation method of primordial follicles provided by the invention adopts an ovary in-situ injection method to inject the preparation into an ovary.

The ovary in-situ injection method comprises the following steps: after the medicine and the Matrigel are fully mixed on ice, a small opening is formed in the position of the ovary on the back of a mouse after the mouse is anesthetized, the ovary is found and clamped out by using forceps and placed on the latex pad, the Matrigel mixed with the medicine is injected at the position of the ovarian cyst, the ovary is moved back to the abdominal cavity after the injection is finished, and the wound is sutured.

The invention provides application of a small molecule drug dbcAMP in preparation of a primordial follicle activation preparation, and experiments show that dbcAMP promotes primordial follicle activation by improving phosphorylation level of mTOR or PI3K/AKT signal pathway key protein, so that the number of primary follicles is increased.

Unless otherwise specified, the raw materials, reagents, consumables and instruments used in the present invention are all common commercial products and are all commercially available.

The invention is further illustrated by the following examples:

example 1

1. Experimental materials:

2-day old female ICR mice, 23-day old female ICR mice.

2. Experimental reagent:

reagent A: the ovary basic culture medium has the specific formula shown in table 1, and the preparation process needs to be carried out aseptically.

TABLE 1 ovarian basal medium formulation

DMEM	0.5g
		F12	0.53g
HEPES	0.357g
		NaHCO3	0.2438g
Penicillin	0.006g
		Streptomycin	0.005g
Ultrapure water	The volume is up to 100mL

And (3) reagent B: 10mM dbcAMP (ovary basal medium as solvent)

And (3) reagent C: sterile PBS

And (3) reagent D: matrigel

3. Procedure of experiment

(1) Sequentially adding 3mL of reagent A and 3 mu L of reagent B into a 6-well plate, placing a cell culture membrane in each well, and then placing the well in a cell culture box for preheating at 37 ℃;

(2) and (3) separating the ovaries of the mice aged 2 days in the reagent C, transferring the ovaries to the culture membrane in the step (1), putting the ovaries into a cell culture box for culture, and activating primordial follicles in vitro. The phosphorylated forms of mTOR, AKT, FOXO3a, rpS6(p-mTOR, p-AKT, p-FOXO3a, p-rpS6), as well as the levels of their total protein, were collected and tested one day later in mouse ovarian tissue (results are shown in figure 1);

(3) collecting ovarian tissues after 3-7 days of ovaries of 2-day-old mice are cultured in vitro, slicing to observe the development condition of each stage of follicles and counting the number of the follicles (the results are shown in figure 2, table 3 and table 4);

TABLE 2 influence of dbcAMP culture concentration on the number of growing follicles and the total number of follicles (unit: pieces)

TABLE 3dbcAMP Effect on Total follicular count (unit: individual)

TABLE 4dbcAMP Effect on the number of growing follicles (Unit: individual)

(4) Ovaries of 23-day-old mice were injected in situ with ovary of Matrigel alone on the left side as a control group and with Matrigel mixed with dbcAMP on the right side (content of dbcAMP in Matrigel: 10. mu. mol/L) as a treatment group, and ovaries were collected two weeks after surgery and sectioned to observe development of follicles at each stage and count the number of follicles (results are shown in FIG. 3 and Table 5).

TABLE 5 Effect of in situ injection of Matrigel or dbcAMP on follicle activation

The results show that: 1. the addition of dbcAMP to the ovaries of mice cultured in vitro promotes the activation of primordial follicles present therein. Further research shows that dbcAMP promotes the in vitro activation of primordial follicles of mice by activating mTOR and a PI3K/AKT signaling pathway; 2.dbcAMP promotes primordial follicle activation in vivo by in situ ovarian injection. Therefore, dbcAMP can be used as a new safe and effective drug-activating target for clinically activating primordial follicles in vitro, and is helpful for further improving the IVA technology by stimulating primordial follicles under a simulated physiological state in the future.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Use of dbcAMP in the preparation of an mTOR agonist and/or an agonist of a key protein of the PI3K/AKT signaling pathway.
Use of dbcAMP in the preparation of a formulation to increase the phosphorylation level of a key protein of the mTOR and/or PI3K/AKT signalling pathway.
3. The use of claim 1 or 2, wherein the mTOR and/or PI3K/AKT signaling pathway key protein is derived from ovary.
Application of dbcAMP in preparation of primordial follicle in-vitro activation drugs.
Use of dbcAMP for the preparation of a medicament for promoting the conversion of primordial follicles to primary follicles.
Use of dbcAMP in the manufacture of a medicament for assisted reproduction, including assisted pregnancy.
7. The medicament for in vitro primordial follicle activation is characterized by comprising dbcAMP and acceptable auxiliary materials and/or auxiliary agents.
8. The medicament of claim 7, wherein the adjuvant and/or adjuvant comprises Matrigel.
9. The medicament of claim 7 or 8, wherein the amount of dbcAMP comprises 1 to 10 μmol/L.
10. The in-vitro activation method of primordial follicles for non-disease diagnosis and treatment purposes is characterized in that ovarian tissues are taken to be cultured in an in-vitro activation reagent;

the in vitro activating agent comprises dbcAMP.